Molecularly targeted imaging agents for the detection, staging, and monitoring of prostate cancer are urgently needed. Prostate Membrane Specific Antigen (PSMA) is a membrane glycoprotein expressed by all prostate cancer. In Phase I of this contract, we reengineered a humanized PSMA-specific antibody to provide a nanoscale biological targeting reagent. Minibodies (~8-10 nm) are significantly smaller than antibodies, yet exhibit dramatically improved properties for in vivo delivery and imaging: full binding combined with rapid in vivo kinetics. Following radiolabeling with I-124 or Cu-64, PSMA-specific minibodies demonstrated excellent biodistribution and microPET imaging properties in a preclinical model of prostate cancer. In Phase II we propose to (1) Optimize labeling conditions for the huJ591 minibody using both residualizing and non-residualizing radiolabels and formally compare biodistribution and imaging performance, in order to finalize the radiolabeling strategy for huJ591 minibody use clinically. (2) Perform a scale-down production run and conduct testing and toxicity studies required for regulatory filing. Public health relevance: A novel, nanoscale imaging agent has been developed for the specific detection of prostate cancer in patients with the goal of commercialization. Success of these PSMA-specific minibodies will establish important groundwork for using this class of engineered biomolecules for additional in vivo nanotargeting strategies.